The present invention relates to an electrochemical immunoassay which can be used for detecting the presence of analytes such as digoxin, theophylline, creatine kinase (CKMB) and human chorionic gonadotrophin (HC). The improvement of this invention involves the use of a complex comprising an electroactive molecule, a carrier molecule and the analyte of interest, in a homogeneous immunoassay.
Electrochemical detection methods are widely employed in the clinical diagnostic market since these methods are relatively inexpensive and simple to use. Heterogeneous electrochemical immunoassay systems frequently require a high degree of operator involvement, due to extensive washing steps and multiple reagent additions, when sensitivities of better than 10.sup.-6 M are desired. Even this level of sensitivity is insufficient for the detection of smaller analytes of clinical significance, such as digoxin, and the detection of most larger analytes such as CKMB and HCG. For instance, digoxin, which is a cardiac glycoside widely used for treating congestive heart failure and other acute cardiac conditions, is a potent drug having a therapeutic effect at concentrations as low as 1 nmol/L with a small therapeutic index. However, digoxin can also be toxic at low concentrations, with the dosage required to produce therapeutic effects and the sensitivity to toxicity being dependent on the particular patient undergoing treatment. Rapid detection of digoxin in the nanomolar range is required for treating cardiac patients, and any useful detection method for digoxin must be capable of measuring nanomolar concentrations with a rapid response time. These problems could be solved by employing a homogeneous system of higher sensitivity, but to date the development of such a system has been elusive. The difficulty in developing such systems based on nonenzymatic electroactive labelling for electrochemical immunoassays is described by W. R. Heineman et al in Methods of Biochemical Analysis, 32, pages 345-393 (1986).
The use of homogeneous electrochemical immunoassays for detecting the presence of theophylline has been reported in M. Haga et al in Analytical Biochemistry, 118, pages 286-293 (1981), using a liposome immunosensor employing liposomes which contain entrapped enzymes whose activity is directly proportional to the lysis of the liposome and inversely proportional to the concentration of free antigen in the sample. When a current pulse is introduced into the sample, the enzyme catalyzes the depletion of oxygen which is detected by an oxygen electrode, and the current registered. The incorporation of electroactive molecules within liposomes is reported in R. M. Kannuck et al in Analytical Chemistry, 60, pages 142-147 (1988), which describes the encapsulation of potassium ferrocyanide within liposomes for signal amplification. The liposome binds with an antibody present in the sample, releasing the encapsulated potassium ferrocyanide, which transmits a charge to the electrode surface. Electroactive liposome technology, while of theoretical interest, is difficult to utilize in practice and can produce false readings due to the instability of the liposomes employed in such systems.
The use of both heterogeneous and homogeneous immunoassay systems involving electroactive enzyme complexes is also described in G. A. Robinson et al, Journal of Immunoassay, 7, pages 1-15 (1986) and in European Patent Application No. 85303367.8, filed May 13, 1985. This technology is directed to the detection of thyroxine by employing a conjugate of thyroxine and ferrocene monocarboxylic acid. The conjugate functions as an electron transfer mediator between an oxido-reductase enzyme, such as glucose oxidase, and an electrode. The ability of the conjugate to function as an electron transfer mediator is impaired by the presence of an anti-thyroxine antibody which binds to the conjugate reducing the current flow to the electrode. In the homogeneous mode, all of the necessary components of system including the conjugate, antibody, thyroxine, glucose oxidase and glucose are initially present or added to the sample. Alternatively, the heterogeneous mode contains the enzyme and antibody which are co-immobilized on the electrode. This system has the disadvantage of being subject to interference, such as interference from oxygen which is present and not purged from the system, or the production of hydrogen peroxide as a side reaction product. Moreover, the additional requirement of enzyme reaction and diffusion necessitates an increased response time for this system, as well as the requirement for measuring both the baseline activity, and the activity level after the additional of sample.
Similarly, both H. M. Eggers et al, Clinical Chemistry, 28, pages 1848-1851 (1982), and T. T. Ngo et al, Applied Biochemistry and Biotechnology, 11, pages 63-70 (1984), describe electrochemical immunoassay techniques for detecting the presence in samples of NADH and DNP-aminocaprodoic acid, respectively. Both of these methods have many of the same disadvantages as the methods discussed previously, and in addition sensitivities of only 10.sup.-6 M are thought to be possible using these approaches.
Chemically-modified enzymes which are capable of directly participating in oxidation/reduction reactions are described by Y. Degani and A. Heller in The Journal of Physical Chemistry, 91, pages 1285-1289 (1986), Journal of the American Chemical Society, 110, pages 2615-2620 (1988), and in European Patent Application No. 88300814.6, filed Feb. 1, 1988. The technology described in these publications involves the reaction of enzymes such as glucose oxidase and D-amino acid oxidase with ferrocene carboxylic acid to form a complex which contains a redox center. The complex is capable of direct electrical interaction with an electrode. Alternatively, the tyrosine groups of glucose oxidase can be transformed into electrochemically active groups, such as DOPA groups, permitting the same interaction. The presence of glucose in a sample is determined by directly measuring the current at the electrode due to the reaction of glucose with the modified enzyme.
As pointed out in European Patent Application No. 88300814.6, the electrolytic modification of an enzyme to incorporate a redox center or couple, while theoretically desirable is usually difficult to achieve. Attempted modifications of enzymes can readily lead to deactivation, such as the attempted modification of carboxy groups in bovine carboxypeptidase A with N-ethyl-5-phenylisoxazolium-3'sulphonate. As indicated in the European patent application, the presence of the redox center in the modified enzyme is believed to be critical since the redox center must be close enough to the electrode to permit electron transfer, while sufficiently removed from the chemical center to avoid deactivation of the enzyme. It will be appreciated that in practice this is a difficult condition to achieve.